Biology Reference
In-Depth Information
Subsequently, the mismatches can be identified with the voltam-
mogram peak potentials of their nanocrystal-mononucleotide tags
[57].
7.3.8
Metal-Ion Amplified Sensor
For practical application, an ideal biosensor must be as straightfor-
wardaspossiblewithleastnumberofsynthesisandanalyticalsteps.
Kraatz and coworkers introduced a simple, label-free and sensitive
electrochemical sensor for single nucleotide mismatch detection.
This approach relies on the diffusive property of the negatively
charged redox probe [Fe(CN)
6
]
3
−
/
4
−
and its interplay with matched
and mismatched DNA films.
Again, the charge transfer resistance
R
ct
for electron transfer
from the solution based anionic redox probe to the transducer
surface is used as a quantifiable measure and is evaluated in the
presenceandabsenceofZn
2
+
usingEISasindicatedinFig.7.13.The
presence of Zn
2
+
in the electrochemical experiment is significant
in that it influences the ability of the [Fe(CN)
6
]
3
−
/
4
−
to diffuse into
the DNA film. In the presence of Zn
2
+
the metal ion will interact
with the phosphate backbone, lowering the electrostatic repulsion
with the anionic redox probe, resulting in a lower charge transfer
resistance. In addition, there are significant differences between
the
R
ct
for matched and mismatched DNA films. Presumably this
is due to differences in packing within the film. Generally, for a
mismatched film, the
R
ct
value will be lower since the mismatched
film will be less densely packed, allowing a better penetration of
the redox probe into the film. Differences in
R
ct
are evaluated in
the presence and absence of Zn
2
+
and in the presence of absence
of a mismatch [88]. This approach allows the detection of single
nucleotide mismatches down to 10 fM level. The method is tolerant
to protein contaminations and also to heterozygote DNA mixtures.
In the absence of Zn
2
+
, the mismatch detection limit is in the
order of 100 nM [89, 90]. The sensitivity produced by metal ions
in ds-DNA film was further confirmed by K'Owino
et al.
[91]
who showed that the addition of Ag
+
to a ds-DNA film gives a
stronger electrochemical response compared to the response for
a ss-DNA film. The simple label-free approach described shows a
